BMC Genomics
BioMed Central
Open Access
Methodology article
Spotted cotton oligonucleotide microarrays for gene expression
analysis
Joshua A Udall*1, Lex E Flagel2, Foo Cheung3, Andrew W Woodward4,
Ran Hovav2, Ryan A Rapp2, Jordan M Swanson2, Jinsuk J Lee4,
Alan R Gingle5, Dan Nettleton6, Christopher D Town3, Z Jeffrey Chen4 and
Jonathan F Wendel2
Address: 1Department of Plant and Animal Sciences, Brigham Young University, Provo, UT, 84062, USA, 2Department of Ecology, Evolution, and
Organismal Biology, Iowa State University, Ames, IA, 50011, USA, 3The Institute for Genomic Research, A Division of the J. Craig Venter Institute,
9712 Medical Center Drive, Rockville MD 20850 USA, 4Section of Molecular Cell and Developmental Biology and Institute for Cellular and
Molecular Biology, University of Texas, Austin, TX, 78712, USA, 5Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia,
30602, USA and 6Department of Statistics, Iowa State University, Ames, IA, 50011, USA
Email: Joshua A Udall* - jaudall@byu.edu; Lex E Flagel - flagel@iastate.edu; Foo Cheung - fcheung@tigr.org;
Andrew W Woodward - woodward@mail.utexas.edu; Ran Hovav - ran@iastate.edu; Ryan A Rapp - rrapp@iastate.edu;
Jordan M Swanson - swansonj@email.arizona.edu; Jinsuk J Lee - jenny-lee@mail.utexas.edu; Alan R Gingle - agingle@uga.edu;
Dan Nettleton - dnett@iastate.edu; Christopher D Town - cdtown@tigr.org; Z Jeffrey Chen - zjchen@mail.utexas.edu;
Jonathan F Wendel - jfw@iastate.edu
* Corresponding author
Published: 27 March 2007
BMC Genomics 2007, 8:81
doi:10.1186/1471-2164-8-81
Received: 24 October 2006
Accepted: 27 March 2007
This article is available from: http://www.biomedcentral.com/1471-2164/8/81
© 2007 Udall et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Microarrays offer a powerful tool for diverse applications plant biology and crop
improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on
three Gossypium species. Using these assemblies as templates, we describe the design and creation
and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated
species.
Results: Synthetic oligonucleotide probes were generated from exemplar sequences of a global
assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many
different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included
on the arrays, optimized to target the diversity of the transcriptome and previously studied cotton
genes, transcription factors, and genes with homology to Arabidopsis. A small portion of the
oligonucleotides target unidentified protein coding sequences, thereby providing an element of
gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA
libraries, the microarray has direct application for analysis of the fiber transcriptome. To illustrate
the utility of the microarray, we hybridized labeled bud and leaf cDNAs from G. hirsutum and
demonstrate technical consistency of results.
Conclusion: The cotton oligonucleotide microarray provides a reproducible platform for
transcription profiling in cotton, and is made publicly available through http://cottonevolution.info.
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Background
Gossypium contains nine different genome groups comprising approximately 50 species whose phylogenetic relationships have been well-studied [1]. The A-, D-, and ADgenome groups have received special attention, as four
different species [Gossypium herbaceum (A1), Gossypium
arboreum (A2), Gossypium hirsutum (AD1) and Gossypium
barbadense (AD2)] have been domesticated for their abundant seed trichomes. These species collectively provide the
foundation for the textile industry worldwide, with most
cotton today deriving from G. hirsutum, or upland cotton.
Relationships among genome groups have been quantified in several studies, and the closest living relatives of
the diploid genome donors to allopolyploid cotton have
been identified [1-5]. The diploid donor of the allopolyploid AT genome [where the T subscript indicates the A
genome in the tetraploid (AD) nucleus], was a species
much like modern G. arboreum or G. herbaceum, whereas
the allopolyploid DT genome is derived from a progenitor
similar to modern G. raimondii. These well-established
relationships provide a phylogenetic framework to investigate the evolution of gene expression both in terms of
domesticated fiber production and polyploidy.
Microarrays are a powerful method to simultaneously
measure relative expression levels for thousands of genes
and they may be composed of cDNA inserts, short oligonucleotides, or long oligonucleotides. The advantages and
disadvantages of each of these probe types have been
extensively reviewed [6-8] We chose to create a long oligonucleotide microarray for cotton because of its low manufacturing cost, flexibility in design, homogeneous
melting temperatures (Tm), and relative ease of adding
probes. A small EST assembly (~45,000 ESTs) was previously used to generate oligonucleotide probes for cotton
fiber [9]. A larger scale EST assembly (> 150,000 ESTs) was
recently produced as a community-wide effort by cotton
researchers [10]. Subsequent additions of cotton ESTs to
Genbank (> 210,000 ESTs) have been compiled into a
large EST assembly [TIGR Cotton Gene Index 8, (CGI8)]
[11]. These two assemblies constitute nearly all of the
known genic sequence from cotton. Similar large-scale
EST assemblies have been successfully used to design oligonucleotide microarrays for functional genomics investigations in model plants (e.g. Arabidopsis[12] and rice[13])
and non-model plants (e.g. maize[14] and tomato[15]).
Here we describe the design and creation and of a publicly
available oligonucleotide microarray for cotton. Synthetic
oligonucleotide probes were generated from the
sequences of two different assemblies of cotton ESTs representing more than 50 different cDNA libraries and
many different tissue types and tissue treatments [10,11]
To illustrate their utility on printed microarray slides, we
hybridized labeled bud and leaf cDNAs from G. hirsutum,
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and demonstrate technical consistency of results. As many
of these EST sequences were derived from a fiber-specific
cDNA library, this array also has direct application for
analysis of the fiber transcriptome.
Results and discussion
Microarray design
We created an oligonucleotide microarray for cotton using
fiber genes in Genbank, a reported EST assembly of
>150,000 ESTs [10], and a recent assembly of >210,000
ESTs [11] as templates for probe design. From these
sequences, we designed three sets of oligonucleotide
probes (1,154, 12,006, and 9,629, respectively) and
included all three sets (22,787 total oligonucleotides) on
a single, publicly available microarray [16]. The first set of
1,154 oligonucleotide probes was designed from Cotton
ESTs with homology to Arabidopsis genes with roles as regulators of chromatin, transcription, cell wall biosynthesis,
and cell cycle [17].
The second set of oligonucleotides probes was designed
from an exemplar sequence set [10] using Picky v1.0 [18].
An exemplar sequence refers to an example gene (i.e., the
longest) chosen from a clustered set of unigenes by singlelinkage clustering with BLASTN [10]. Picky prioritized the
unique sequence of the identified oligonucleotide probes
while maintaining a uniform probe-target melting temperature. 12,006 oligonucleotides (66 bp average length;
3.5 s.d.) with a relatively small range of melting temperatures (Tm, 78.33 ± 1.40 s.d.) were selected from a large list
of candidate probes. This list of targeted genes includes
genes requested by members of cotton research community, a large number of transcription factors, and several
thousand genes that had homology to Arabidopsis genes
(Table 1) [see Additional file 1].
The third set ofoligonucleotides probes was designed
from CGI8 [11] that contained 55,673 unique sequences
using Picky v2.0. Where possible, identical Picky parameters were used to design the 3rd oligonucleotide set as the
2nd set. Probes that targeted the same genes as in the first
two probe sets were excluded from further analysis. In
total, 9,629 additional oligonucleotides probeswere generated (66 bp average length, 3.6 s.d.; 76.82 Tm, s.d. 1.93)
and added to the previous 2 probe sets.
Two essential considerations of microarray quality
include the number of targeted genes and the broad utility
of the microarray for specific tissues or treatments.
Regarding the first consideration, the 22,778 genes
described here include perhaps 46–60% of the total genic
diversity, given that the total number of genes in the cotton genome may be approximately 40,000–50,000
[19,20], Indeed, 44% and 40% of the oligonucleotides
were designed from singletons from the first [10] and sec-
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Table 1: Oligonucleotide probes were designed separately from three different sets of ESTs.
Types of probes
Arabidopsis matches5
Singletons
Transcription factors (TF)6
GO Biological Process7
GO Molecular Function8
PFAM9
Total number of oligos
1st oligo set1
2nd oligo set2
3rd oligo set3
Totals4
866
na
230
46
184
na
7,419
5,280
2,223
464
1,759
471
4,031
3,852
677
126
551
na
12,316
9,132
3,130
636
2,494
471
1,154
12,006
9,629
22,789
1Oligonucleotides (oligos) were designed at Texas A&M University in the former lab of Dr. Chen [17]. 2Oligos designed at Iowa State University
from a global assembly of ESTs [10]. 3Oligos designed by The Institute for Genomic Research (TIGR) from the Cotton Gene Index 8 [11]. 4Column
totals are close approximations since each oligo set was designed separately. 5Arabidopsis matches are based on oligo WUBLASTX hits to the TAIR
Arabidopsis protein dataset and parsed for 50 aa length and 50% percent identity. 6Transcription factor sub-total may be an over-estimate as
Biological Processes and Molecular Function are not mutually exclusive categories. 7Biological Process = transcription. 8Molecular Function =
nucleic acid binding activity, nucleotide binding activity, RNA and DNA binding activity, and transcription factor activity. 9Putative PFAM TF
identified in the 2nd oligo set were in addition to those annotated by gene ontology.
ond [11] assemblies, respectively. However, some probes
were not completely target-specific, perhaps due to imperfect EST assemblies and due to separate sets of probes
designed from different assemblies. Approximately 1,800
of potential non-specific homologies were identified in
the recent Gene Index of 55,673 unigenes [11] using
vmatch [21] with a ~95% sequence percent identity
threshold. Nevertheless, most of the oligonucleotides had
a single target, as designed [see Additional file 2].
Regarding the second consideration, these microarray
probes have a broad utility for specific tissues or treatments. For example, a detailed analysis of the second
probe set revealed that ~7,300 probes represented genes
expressed in specific tissues or under specific conditions
[see Additional file 3]. The number of specifically
expressed genes was determined by summing the number
of contigs mostly composed of ESTs from a single library
(90%) and the corresponding singletons. In total, 56% of
the 2nd oligonucleotide set represent genes from a specific
library; however, a large number of those genes (24%) are
from the two G. raimondii libraries that were prepared
from heterogeneous tissues and which were more deeply
sampled that most of the other cotton cDNA libraries.
More than 1,000 oligonucleotides appear to target genes
found only in a 7–10 days post-anthesis fiber library [9],
and 733 appear to target transcripts uniquely identified
following cyclohexamide treatment of ovules [22]. These
two considerations suggest that the oligonucleotides
selected for the cotton oligonucleotide microarray have a
broad diagnostic utility while potentially targeting tissuespecific transcripts expressed under a variety of conditions. The sequences and annotations of all the probes are
publicly available via a web-based query [16] or by
request.
Microarray hybridizations
Many potential sources of error can have a large impact on
microarray experiments, such as inconsistency among
multiple RNA extractions, reverse transcription, RNA
amplification, and labeling, as well as different levels of
background noise for each microarray. Many of these
sources of error can be resolved by appropriate experimental design [23,24]. and careful laboratory technique;
however, the quality of the microarray must often be
assumed and often is not under the control of the investigator. We investigated the oligonucleotide performance
within the first version of the cotton microarray [Gene
Expression Omnibus (GEO) database: GPL4305] containing the 1st and 2nd replicated spots printed for each probe
in these two sets. We found a low level of within-microarray variation, reproducible 'self vs. self' hybridizations
with bud tissue, and reproducible expression differences
between bud and leaf treatments. The results from the first
version of the cotton microarray suggest that the current
version with 9,629 additional probes will provide an
robust, reproducible platform for transcription profiling
in cotton.
Variation within microarrays between the two replicate
features was estimated for each oligonucleotide probe, as
a consequence of including two microarray features for
each oligonucleotide probe in two separate sections of the
microarray. The average log-adjusted difference between
two replicated features was 0.03 (s.d. 0.69), 0.02 (s.d.
0.80), and 0.02 (s.d. 0.83) for replications 1, 2, and 3,
respectively (Figure 1A). The slightly positive value of the
average spot differences suggests that the first pin-touch
on the microarray deposited slightly more oligonucleotide probe in a slightly larger spot on the slide than the
second pin-touch.
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Figure
A
reproducible
1
platform for transcription profiling using cotton microarrays
A reproducible platform for transcription profiling using cotton microarrays. A) Differences detected between
duplicated spots plotted by their mean expression value from replicate 1 of the experiment. Each plot is hexagonally-binned to
uncover the density component that is otherwise saturated by a cloud of data points. The difference of the log-adjusted,
median centered duplicate spots is measured on the horizontal axis and the mean value of the same duplicated spots is measured on the vertical (values in grey, scale bars on left vertical axis). Most duplicated spots deviated very little, though genes
with lower expression values tended to deviate more. Replication of the three treatment loop design indicated only minor
detectable differences between duplicated spots. Nearly identical results were found for the other two replicates. B) Correlation of normalized, log-adjusted fluoresce intensity values for bud (Cy3) × bud' (Cy5) for the first microarray of the first replication. A 45° angle line has been overlaid to illustrate the expected 1:1 ratio of spot intensity. In this case, Cy3 labeled aRNA
had higher intensity values on average; however, the effect of dye was removed from the contrast of differential expression in
our analysis by including a dye component into our analytical model through dye swaps. C) Correlation of t-test p-values.
Gene-specific tests for differential expression between bud and leaf and between bud' and leaf were conducted as described in
Materials and Methods. The p-values from the bud' vs. leaf tests are plotted against the p-values from the bud vs. leaf tests on a
negative natural log scale. The points in the upper right quadrant of the picture correspond to the genes with the smallest pvalues. The points are scattered tightly around the 45 degree line, indicating that that p-values for the most significant genes
were very similar according to both comparisons.
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We also demonstrated the reproducibility of the microarray by quantifying the amount of variation for these
newly constructed cotton microarrays between two identical pooled 'treatments' of bud RNA (bud and bud'). On
the individual microarrays, there was a high degree of correlation between the Cy3-labeled bud RNA and the Cy5labeled bud RNA (r = 0.92 data not shown). When searching for expression differences between bud and bud' RNA
pools, no differences were detected at a relatively liberal
false discovery rate of 20% (Table 2).
Finally, if we test for expression differences between the
leaf RNA pool and each of the bud RNA pools (bud and
bud'), many genes were found to be consistently differentially expressed between bud vs. leaf and bud' vs. leaf, as
seen by the high correlation among the most significant pvalues (Figure 1B). If our goal was to describe differentially expressed genes between leaf and bud, we would
have a large list of putatively, differentially expressed
genes (Table 2); however, in this particular case, we can
only claim that these genes were differentially expressed
between the two amplified RNA samples considered in
our experiment. We intentionally did not include biological replicates because our interest was in quantifying
technical variation. Biological replication would be necessary to conclude that the expression differences are inherent differences between leaf and bud rather than simply
differences between the particular leaf RNA pool and the
particular bud and bud' RNA pools prepared for this
experiment.
Because a global assembly of ESTs was used to design this
first version of the microarray, genes expressed in many
tissues of the plants including cotton fibers, are represented. A total of 1,864 genes were found to be differentially expressed between our leaf RNA pool and our bud
RNA pools (false discovery rate = 0.001; Figure 1C).
(Again note that these are differences between our RNA
pools rather than inherent expression differences between
general leaf and bud tissues.) Of these differences, slightly
less than 10% belonged to set of probes that had no
BLASTX hit (< 1 × 10-20) and were designed solely based
on an ESTScan prediction, thereby providing an element
of functional gene discovery to the microarray. Of the
1,864 hypothetical genes that were differentially
expressed, 602 were identified by probes derived from
cotton fiber cDNA libraries [9]; Ben Burr, unpublished
data; Candace Haigler, unpublished data). This number
did not include probes designed for known cotton genes,
nor other genes that were removed from the analysis (low
expressed fiber genes that may not be detected in leaf or
bud aRNA) that may also represent genes specifically
expressed in fiber tissue.
Conclusion
Here we provide a detailed report of the design of longoligonucleotide microarrays for cotton and illustrate their
technical performance. Proper design of microarray experiments for discovery of gene expression profiles requires
biological replication [24-26] Because our goal was not to
discover nor report novel gene expression profiles in
leaves or buds, we restricted our replications to pools of
technical replicates in this experiment. These cotton
microarrays are publicly available [16], and may continue
to be augmented with additional oligonucleotides
designed from subsequent ESTs assemblies. As a tool for
functional genomics, the future use of these microarrays
may uncover clues to the transcriptional regulation of cotton fiber and other tissues in properly replicated experimental designs.
Methods
Microarray probe selection
A total of 22,787 probes were printed on the cotton oligonucleotide microarray, composed of 12,006 oligonucleotides selected from recently reported contigs and
singletons derived from a global assembly of cotton ESTs
[10], and 9,629 designed against the latest TIGR cotton
transcript assembly (CGI8). We also included 1,154 oligonucleotides designed in the Z. J. Chen laboratory based on
previously available sequence data [27].
Similar probe design strategies were used for all threes sets
of probes. Lee et al. [17] provided a description for the
design first set of oligonucleotides. The second and third
set of oligonucleotides were designed from the EST exemplar sequences [10] and the TIGR Gene Index [11] with
Picky (v 1.0) [18] and Picky (2.0), respectively. Only EST
sequences with predicted a protein from ESTScan [28] or
a high protein homology (70% percent identity) to an
Arabidopsis gene were considered candidates for oligonucleotide selection.
Table 2: Number of differentially expressed genes at different levels of false discovery [34].
Comparison
0.001
0.010
0.050
0.100
0.200
0.300
bud – bud'
bud – leaf
bud' – leaf
0
2167
2232
0
4,506
4,608
0
6,562
6,506
0
7,600
7,641
0
8,933
9,007
20
9,694
9,654
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Three different criteria were invoked in the iterative selection of the microarray probes from the EST assemblies: (1)
candidate probes identified by Picky by their unique
sequences, complexity, and Tm; (2) characterized cotton
mRNAs in GenBank and genes of special interest to the
cotton community; and (3) complementation to previously synthesized probe set(s). Thus, for each oligonucleotide set, candidate long-oligonucleotide probes (60 – 70
mers) were separately generated based on criteria 1 and 2,
then a final list of probes was selected for oligonucleotide
synthesis by cross-checking the new list of probes with
previously synthesized oligonucleotides.
For design of both the second and third probe sets, we
solicited input from the cotton community to identify
genes of interest for microarray probe design. Most
requests were for known cotton genes with sequences in
Genbank, in addition to candidate genes identified in our
EST assembly by high homology to genes characterized in
other organisms. Probes for these genes were designed
with the sequences from the EST assemblies as 'background' to identify the most unique probes possible.
Both transcription factors and genes with little or no
annotation were represented on the cotton microarray.
Based on widespread interest in transcription factor
expression levels, we selected probes targeting genes that
had either a transcription related GO ontology [29], or a
transcript factor domain as predicted by PFAM [30] (Table
1). Genes with little or no annotation represent a gene discovery component to future microarray experiments.
When genes targeted by the 2nd probe second were compared to the Arabidopsis TAIR protein dataset, 1,200 of
them did not have a significant BLASTX hit (< 1e-20) but
they did have a coding frame as predicted by ESTScan
[28].
Oligonucleotide synthesis and microarray printing
Each set of oligonucleotides was synthesized and aliquoted into 3 replicate plates by IDT Technologies (Coralville, IA, USA). An aliquot of 384-well plates from all
three sets of oligonucleotides was hydrated in water then
diluted to the printing concentration with 3× SSC. Positive and negative controls were included on the printed
microarrays. To assess microarray quality, two spots of
each oligo from the same pin-dip were printed in separate
slide sections on Corning epoxy slides at the Washington
University Microarray Core facility using a locally constructed linear servo arrayer (after the DeRisi model [31])
creating the first version of the cotton oligonucleotide
microarray [GEO: GPL4305]. After printing, slides were
allowed to dry in 50–70% humidity for 12–16 hrs
(~25°C) and cross-linked at 150 mJoules. Two slides from
the print batch were checked using SpotCheck (Genetix).
Printed cotton microarrays, and images of each print
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batch are publicly available [16]. Experiments using the
preliminary platform [GEO: GPL4305] or this new platform [GEO: GPL4808] can be found at GEO [32].
RNA extraction
One leaf and one bud (10 – 14 days before anthesis) tissue sample of G. hirsutum cv. Acala Maxxa were collected
from three separate replications of 4 – 8 plants grown in
Horticulture Greenhouse at Iowa State University under
supplemental lighting (16 hr. days). RNA was extracted
from each of the six samples using a modified hot-borate
method [33], quantified, and checked for integrity using a
Bioanalyzer (Agilent, Inc., Palo Alto, CA, USA). Equimolar amounts of RNA (A260) from three separate extractions
were pooled into a single leaf and single bud sample,
respectively.
RNA amplificationand labeling
An indirect labeling procedure of amplified aminoallyl.aRNA (TargetAmp™, Epicentre Biotechnologies,
Madison, WI, USA) was used for one leaf RNA sample and
one bud RNA sample. 0.5 ug of total RNA was used as
starting material for 1 round of aRNA amplification,
resulting in 26 ug and 51 ug of aRNA from leaves and
buds, respectively.
Cy3 and Cy5 dyes (Amersham Biosciences, Pittsburgh,
PA, USA) were coupled to two aliquots of 13 and 16 ug of
both aRNA samples, respectively. The Cy3- and Cy5labeled aRNA probes were purified using the Qiagen RNA
easy Mini kit (Qiagen, Germantown MD, USA). and sufficient incorporation Cy3 (550 nm) and Cye5 (650 nm)
dyes was verified.
Microarray hybridization and image analysis
For microarray hybridization, 300 ng of Cy3 and Cy5
labeled aRNA was used per each slide using the Pronto!™
Plus system protocol (Promega Corporation, Madison
WI, USA) with minor changes as described below. Slides
from each rep (3) were immersed in 200 ml of Pronto
Universal Pre-Soak solution containing 2 ml of liquid
Sodium Borohydride for 20 min at 42°C. Slides were
transferred to fresh containers with Wash Solution 2 at
room temperature for 2 min and then immersed in 200
ml of hybridization buffer (5 × SSC; 0.1 × SDS; BSA 0.1
mg/ml). Slides then were incubated with a fresh Wash
Solution 2 at room temperature for 2 min, and were
washed 2 additional times with Wash Solution 3 at room
temperature for 2 min each. Following immersion in
nuclease-free water, slides were dried by centrifugation at
1,600 g for 3 min. All hybridizations and post-hybridization washes were performed exactly as described in the
Pronto!™ Plus system protocol.
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Microarray images were captured using an arrayWoRx®
Biochip Reader (Applied Precision, Issaquah, WA, USA)
using an exposure of 0.5 sec for each channel (Cy5 and
Cy3) at ~10 um resolution. GenPix® Pro (v 5.1, Molecular
Devices, Sunnyvale, CA, USA) was used to extract the
background-adjusted intensity of each spot. Features that
were 'absent', 'not-found', or that had a negative intensity
after background adjustment were excluded from the
analysis. Data files from this experiment can be found in
GEO data set [GSE5875].
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performed the microarray hybridizations. DN provided
the statistical model and analytical framework. LF, JAU,
and DN analyzed and interpreted the data. ARG customized an internet accessible MIAME-compliant Stanford
Microarray Database for these arrays and created a GEO
microarray platform for this and future experiment submissions. JAU, RAR, and JFW conceived the experiment.
JAU and JFW drafted the manuscript. All authors have
read and approved the manuscript.
Availability and requirements
Experimental design and statistical analysis
Three replications of a three treatment loop design (bud
→ leaf, leaf → bud', and bud' → bud) were hybridized on
nine microarrays, where bud and bud' simply represent
different aliquots of the same aRNA. The signal intensity
data were natural log transformed and median normalized, and the 9,654 genes with complete data were examined for expression differences among the three sample
types (leaf, bud, and bud'). We considered a standard
mixed linear model for the data from any single gene
given by
yijk = μ + δi + τj + sk + eijk,
where yijk denotes the normalized log-scale signal intensity (averaged over duplicate spots) for dye i, sample type
j, and slide k; μ denotes a an intercept parameter; δi
denotes the effect of dye i; τj denotes the effect of sample
type j; sk denotes the random effect of slide k; and eijk
denotes a random error term that is intended to capture
all other sources of variability. (Note that although we
considered a separate model for each gene, we have suppressed a gene subscript on each term to simplify notation.) Here i = 1, 2 (Cy3 and Cy5); j = 1, 2, 3 (bud, bud',
and leaf); and k = 1, ..., 9 (microarray slides 1 – 9). On the
basis of this model, t-tests for differential expression
between each pair of sample types (leaf vs. bud, leaf vs.
bud', and bud vs. bud') were conducted. The 9,654 p-values from each of these comparisons were converted to qvalues using the method of Story and Tibshirani [34].
These q-values were used to identify the number of differentially expressed genes for a given comparison when
controlling the false discovery rate at various levels.
Abbreviations
ESTs: Expressed sequence tags
Authors' contributions
JJW, JJL, and ZJC designed and provided the first set of oligonucleotides. JAU, JMS, JFW, and ZJC designed, analyzed, and provided the second set of oligonucleotides.
FC, AWW, CDT, and ZJC designed and provided the final
set of oligonucleotides. LF and JAU grew the plants and
extracted RNA. RH, LF, and RAR amplified, labeled, and
Project name: The evolutionary genomics of cotton
Project home page: http://cottonevolution.info
Operating system: Platform independent
Programming language: HTML and XML
License: no license required
Additional material
Additional file 1
Composition of the cotton oligonucleotide microarray. 22,789 oligonucleotides were designed from three separate sets of genic sequences from
cotton (See Table 1). The grey boxes represent the total number of probes
in each set. The hatched boxes indicate the number of probes with a putative Arabidopsis hit. The black boxes indicate the number of probes
designed from singletons from their respective assemblies. The remaining
boxes with dotted squares indicate the number of probes targeting transcription factors.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-8-81-S1.pdf]
Additional file 2
Distribution of the number of matches of oligonucleotide probes to the
Cotton Gene Index 8 (CGI8) assembly. All three sets were queried
within the sequences of the CGI8 assembly and only a small number
(1,773) of probes target (>93% percent identity) more than one CGI8
unigene indicating a potential cross-hybridization or an 'over-split' assembly for the targeted gene.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-8-81-S2.pdf]
Additional file 3
Many of the oligonucleotides from the 2nd set were derived from contigs or singletons representing individual libraries (n = 7,319). Library
totals reflect the contigs (respective library's ESTs > 90%) and singletons
used to design the oligonucleotides. The two large G. raimondii libraries
created from heterogeneous seedling and whole flower tissue are not illustrated.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-8-81-S3.pdf]
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Acknowledgements
The work was supported by the grants from the National Science Foundation Plant Genome Research Program (DBI0211700 to J.F.W. and
DBI0624077 to Z.J.C.).
23.
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